One of the hallmarks of age-associated immune dysfunction is the markedly diminished response of T lymphocytes. The dramatic increase in morbidity and mortality from infections is a prime example of a clinical outcome of the declining T cell immune capacity in the elderly. Recent research in this laboratory has demonstrated that peripheral blood from aged donors contains an increased proportion of T cells lacking expression of the CD28 costimulatory molecule, an essential component of the activation pathway. Cultured T cells established from young adult donors which have reached a state of replicative senescence (i.e., absence of ability to proliferate following numerous rounds of cell division) also lose expression of CD28 antigen. CD28- T cells, irrespective of whether they arise in long-term culture following repeated antigenic stimulation or are freshly isolated from peripheral blood, have significant shortening of chromosomal telomere DNA, the best available molecular measure of a cell population's remaining proliferative potential. The proposed studies will use the novel human T cell culture system developed in this laboratory to analyze the molecular and genetic mechanisms responsible for replicative senescence as a tool to identify suitable immunomodulatory strategies for the elderly. These cultures, which are established from cryopreserved mononuclear cells isolated from young healthy donors, provide the unique opportunity to study within a period of months the phenomenon of replicative senescence which occurs in vivo over a span of many years. The following hypotheses will be tested: I. Maintenance of CD28 antigen expression will delay or prevent replicative senescence in T lymphocytes. A second CD28 gene, under control of an inducible promoter, will be introduced into cultured T cells by retroviral-mediated gene transfer to determine the effect on replicative capacity. II. CD28- T cells are insensitive to apoptosis. Therefore they accumulate with age. In the thymus, CD28 engagement is an essential component of thymocyte apoptosis, suggesting that mature peripheral T cells which lack CD28 may be incapable of undergoing apoptosis. The long-term culture will be used as a source of CD28+ and CD28-T cells for analysis of apoptosis using flow cytometric and DNA laddering techniques. III. CD28+ T cells comprise a heterogeneous population with respect to proliferative potential. The loss of CD28 expression at the stage of replicative senescence implies that within the CD28+ T cell population, some cells are further along the path to senescence than others. The long-term cell culture model will be used to explore the dynamics of the loss of CD28 expression in order to identify pre-senescent markers. These basic science studies, though performed entirely on cultured cells, are viewed as "Intervention Development" since the results of the proposed research will help identify potential immunomodulatory strategies aimed at improving T cell immunity to infections in the elderly. These include gene therapy with CD28, augmentation of apoptosis of senescent, non-functional T cells, and identification of additional informative markers for clinical evaluation of immune status in the elderly.